Low-level laser treatment promotes skin wound healing by activating hair follicle stem cells in female mice.

Abstract

The aim of the study was to explore the effect and mechanism of a low-level laser on hair follicle stem cells in full-thickness skin wound healing in mice. Full-thickness skin defects were generated by a 5-mm punch biopsy tool on the backs of depilated C57/BL6N mice, which were randomly divided thereafter into a low-dose laser treatment group (LLLT-Low), a high-dose laser treatment group (LLLT-High), and a control group (control). From the day of modeling to the day before the skin samples were taken, the wound area and wound edge of the mice in the LLLT-Low and LLLT-High groups were irradiated with a laser comb every 24h, and the energy density was 1J/cm2 and 10J/cm2, respectively. The control group was irradiated with an ordinary fluorescent lamp. At 0, 3, 5, 10, and 14days after modeling, pictures of each wound were taken, and the percent wound closure was analyzed. At 3, 5, 10, and 14days after modeling, the samples were observed by hematoxylin and eosin (HE) and immunofluorescence (IF) staining. Whole transcriptome sequencing (RNA-Seq) was performed on the samples on day 10. Gene Ontology (GO) analysis was performed, and the results were validated by Western blot analysis and enzyme-linked immunosorbent assay (ELISA). The analysis of the percent of wound closure showed that healing was accelerated (significantly from 5 to 10days) in the LLLT-Low group, but there was no clear change in the LLLT-High group. HE staining showed that the LLLT-Low group had an increasing number of hair follicles and a tendency to migrate to the center of the wound. There was no significant increase in the number of hair follicles and no obvious migration in the LLLT-High group. Immunofluorescence staining showed that the total number of CK15 + hair follicle stem cells in the LLLT-Low group was higher than that in the control group and LLLT-High group at all time points. The number and farthest migration distance of CK15 + hair follicle stem cells increased significantly with time, and after 5days, they were significantly higher than those in the control group and LLLT-High group. RNA-Seq and Western blot analysis showed that the expression of related genes in hair follicle stem cells, including CK15, in the LLLT-Low group was upregulated. GO analysis and ELISA showed that the expression of many cytokines, represented by IL34, in the LLLT-Low group was upregulated. Low-level laser treatment can promote the proliferation, differentiation, and migration of CK15 + hair follicle stem cells by upregulating the cytokine IL34, thereby promoting skin wound healing in mice.